Antiangiogenic active immunotherapy

ABSTRACT

Application of oligonucleotide and polypeptide sequences of molecules of the family of the vascular permeability factor (VPF), their receptors, and co-receptors, as well as their modifications, in the active immunotherapy of pathologic entities in which course is associated to the increase of angiogenesis. 
     These procedures can be employed in the single or combined therapy for the treatment of cancer and its metastasis, acute and chronic inflammatory processes, infectious diseases, autoimmune diseases, diabetic and newborn retinopathies, organ transplant rejection, macular degeneration, neovascular glaucoma, hemangioma, and angiofibroma, among others.

BACKGROUND OF THE INVENTION

The present invention is related with the field of biotechnology andpharmaceutical industry, in particular with active immunizationemploying as targets molecules related with angiogenesis.

The process of formation of new blood vessels from pre-existent ones iscalled angiogenesis. This event is widely regulated through theequilibrium of pro- and anti-angiogenic factors. Among the diseases inwhich the course has been related with the induction of pro-angiogenicfactors and the formation of new blood vessels in anomalous form are:(a) cancer (both primary tumors and their metastases), (b) acute andchronic inflammatory processes such as asthma, respiratory distress,endometriosis, atherosclerosis, and tissular edema, (c) diseases ofinfectious origin as the Hepatitis, and Kaposi sarcoma, (d) autoimmunediseases as diabetes, psoriasis, rheumatoid arthritis, thyroiditis, and(e) other diseases and states as the diabetic and newborn retinopathies,organ transplant rejection, macular degeneration, neovascular glaucoma,hemangioma, and angiofibroma (Carmelliet P. y Jain R K. Nature 407:249,2000; Kuwano M, et al. Intern Med 40:565, 2001). A potentiallyattractive therapeutic procedure for many of these cases could be basedon the inhibition of the activity of the pro-angiogenic factors, thatstimulate the anomalous formation of blood vessels, via theirneutralization, or that of their receptors, or by eliminating thesources that produces them.

Vascular endothelium growth factors are a family of molecules thatinduce the formation of new vessels specifically and directly (LeungScience 246:1306, 1989; Klagsburn M, Annual Rev Physiol 33:217, 1991).This family includes the vascular permeability factor, also known asvascular endothelium growth factor VPF/VEGF (now denominated VEGF-A),the placenta growth factor PIGF, the platelet derived growth factorsPDGF-A and PDGF-B, and other four new molecules structurally andfunctionally related to VEGF-A designated VEGF-B/VRF, VEGF-C/VRP,VEGD-D/FIGF, and VEGF-E. (Olofsson B et al. PNAS USA 13:2576, 1996;Joukov V et al. EMBO J 15:290, 1996; Yamada Y et al. Genomics 42:483,1997; Ogawa S et al. J Biol Chem 273:31273, 1998).

VEGF-A is a homodimeric glycoprotein formed by two 23-kDa subunits(Ferrara N, et al. Biochem Biophys Res Comun 165:198, 1989), of whichfive monomeric isoforms exist, derived from the differential splicing ofthe same RNA. These include two isoforms that remain attach to thecellular membrane (VEGF 189 and VEGF 206), and three of soluble nature(VEGF 121, VEGF 145, and VEGF 165). VEGF 165 is the most abundantisoform in mammal tissues, except for lung and heart, where VEGF 189predominates (Neufeld G et al. Canc Met Rev 15:153, 1995), and inplacenta, where VEGF 121 expression prevails (Shibuya M A et al. AdvCanc Res 67:281, 1995).

VEGF-A is the most studied and characterized protein of this family, andits alteration has been described in a larger number of diseases. Itsover-expression is associated to tumors of different origin andlocalization, and their metastasis (Grunstein J et al. Cancer Res59:1592, 1999), chronic inflammatory processes as ulcerative colitis andCrohn's disease (Kanazawa S, et al. Am J Gastroenterol 96:822, 2001),psoriasis (Detmar M, et al. J Exp Med 180:1141, 1994), respiratorydistress (Thickett D R et al. Am J Respir Crit Care Med 164:1601, 2001),atherosclerosis (Celletti F L et al. Nat Med 7:425, 2001; Couffinhal Tet al. Am J Pathol 150:1653, 1997), endometriosis (McLaren J. Hum ReprodUpdate 6:45, 200), asthma (Hoshino M, et al. J Allergy Clin Immunol107:295, 2001), rheumatoid arthritis and osteoarthritis (Pufe T et al. JRheumatol 28:1482, 2001), thyroiditis (Nagura S et al. Hum Pathol 32:10,2001), diabetic and newborn retinopathies (Murata T et al. Lab Invest74:819, 1996; Reynolds J D. Paediatr Drugs 3:263, 2001), maculardegeneration and glaucoma (Wells J A et al. Br J Opthalmol 80:363, 1996;Tripathi R C et al. Opthalmology 105:232, 1998), tissular edema (Kaner RJ et al Am J Respir Cell Mol Biol. 22:640 2000; Ferrara N Endocrinol Rev13:18, 1992), obesity (Tonello C et al. FEBS Left 442:167, 1999),hemangiomas (Wizigmann S y Plate K H Histol Histopathol 11:1049, 1996),in the synovial fluid of patients with inflammatory arthropathies(Bottomley M J et al Clin Exp Immunol 119:182, 2000), and associated totransplant rejection (Vasir B, et al. Transplantation 71:924, 2001). Inthe particular case of tumors, the cells expressing the three basicisoforms of VEGF-A: 121, 165, and 189, are the ones that grow faster invivo; while in final stages most tumors limit expression to the VEGF 165isoform, or, in its absence, to a combination of 121 and 189 that farfrom being additive, evidences a cooperation that strengthens the tumorvascular network (Grunstein J. Mol. Cell Biol 20:7282, 2000).

PIGF, described in 1991, is not able to induce endothelial proliferationin its homodimeric form (Maglione D et al. Proc Natl Acad Sci USA88:9267, 1991, DiSalvo J et al. J Biol Chem 270:7717, 1995). With PIGFup-regulation, and with it, of the signal transduced via VEGFR-1, theendothelial cells amplify their responses to VEGF during the change tothe angiogenic phenotype associated to certain pathologies (Carmeliet Pet al. Nat Med 7:575, 2001). PIGF expression has been related to thevascularization of human meningioma and glioma (Nomura M et al. JNeurooncol 40:123, 1998). This molecule forms heterodimers with VEGF165, with pro-angiogenic activity, and their over-expression has beendescribed in the conditioned media of some tumor cell lines (Cao Y etal. J Biol Chem 271:3154, 1996), and associated to the evolution ofrheumatoid arthritis and to primary inflammatory arthropathies, ingeneral (Bottomley M J et al. Clin Exp Immunol 119:182, 2000).

The over-expression of the rest of the members of the VEGF family, lessstudied, is also associated to a number of pathologies. VEGF-B has beenrelated to breast, ovary, and kidney tumors, and to melanomas andfibrosarcomas (Sowter H M, et al. Lab. Invest. 77:607, 1997; Salven PAm. J. Pathol. 153:103, 1998, Gunningham S P et al. Cancer Res 61:3206,2001). The differential expression of the VEGF-B 167 isoform in vitrohas been reported in tumor cells of diverse origin (Li X, et al. GrowthFactors 19:49, 2001). VEGF-C and VEGF-D are involved in the regulationof lymphatic vessels formation (Joukov V. et al EMBO J. 15: 290, 1996),and VEGF-C over-expression is associated to tissular edemas, to tumorsof the breast, lung, head and neck, esophagus, and stomach, lymphomas,prostate cancer, and metastatic nodes (Kajita T, et al. Br J Cancer85:255, 2001; Kitadi Y, et al Int J Cancer 93:662, 2001; Hashimoto I, etal. Br J Cancer 85:93, 2001; Kinoshita J, et al. Breast Cancer Res Treat66:159, 2001; Ueda M, et al. Gynecol Oncol 82:162, 2001; Salven P Am. J.Pathol. 153:103, 1998; O-Charoenrat P et al. Cancer 92:556, 2001). Inthe case of VEGF-D, its over-expression by tumor cells is related to anin vivo increase of lymphatic vasculature in the tumors and the increaseof metastasis in lymphatic nodes (Stacker S A, et al. Nat Med 7:186,2001; Marconcini L et al. Proc Natl Acad Sci USA 96:9671, 1999).

The alterations on endothelial cell function induced by the molecules ofthe VEGF family are mediated by their binding to cell receptors of thetype tyrosine kinase class 3, that so far include: VEGFR1 (Flt1), VEGFR2(KDR/Flk1), and VEGFR3 (Flt4) (Kaipainen A J. Exp. Med. 178:2077, 1993).The N-terminal domain 2 has been identified as responsible of thebinding to the ligands, favoring the phosphorilation of thecytoplasmatic domain and transduction of the signal (Davis-Smyth T et alEMBO 15:4919, 1996).

Ligands identified for VEGFR1 include VEGF-A, PIGF, and VEGF-B, indecreasing order of affinity (Shibuya M Int J Biochem Cell Biol 33: 409,2001). In endothelial cells, this receptor captures the circulating VEGF(Gille H et al EMBO J. 19:4064, 2000). The binding of VEGF-A to theVEGFR1 expressed in cells of the hematopoyetic lineage affectssignificantly the activation of transcriptional factor NFκB in theprecursors of dendritic cells, and in B and T lymphocytes. This lastinteraction is relevant in the in vivo establishment of an unfavorableimmunologic balance, where dendritic cells maturation and the fractionof T lymphocytes are reduced, a phenomenon observed on immunosuppressedpatients and in particular, with cancer (Dikov M M et al Canc Res61:2015, 2001; Gabrilovich D et al. Blood 92:4150, 1998).Over-expression of VEGFR1 has been related with psoriasis, endometrialcancer and hepatocellular carcinoma (Detmar M, et al. J Exp Med180:1141, 1994; Ng IO Am J Clin Patol 116:838, 2001; Yokoyama Y et alGynecol Oncol 77:413, 2000).

The VEGFR2 receptor (KDR/Flk1) mediates the biological effects ofVEGF-A, and also binds VEGF-C and VEGF-D. This receptor is expresseddifferentially on activated endothelium and in some cell lines of tumororigin where it establishes autocrine pathways with the secreted VEGF.Apart from being involved in the already mentioned pathologies that arerelated with the over-expression of its ligands, the over-expression ofVEGFR2 has been related with the progression of endometrial cancer(Giatromanolaki A et al, Cancer 92:2569, 2001), malignant mesothelioma(Strizzi L et al. J Pathol 193:468, 2001), astrocytic neoplasms (CarrollR S et al. Cancer 86:1335, 1999), primary breast cancer (Kranz A et al.Int J Cancer 84:293, 1999), intestinal type gastric cancer (Takahashi Yet al Clin Cancer Res 2:1679, 1996), multiform glioblastoma, anaplasticoligodendroglioma, and necrotic ependimoma (Chan A S et al. Am J SurgPathol 22:816, 1998). Over-expression of KDR has also been associated tothe autosomic disease VHL and to hemangioblastoma (Wizigmann-Voos S etal Cancer Res 55:1358, 1995), to the progression of diabetic retinopathy(Ishibashi T. Jpn J Opthalmol 44:323. 2000) and, in combination withFlt-1 over-expression, to the delayed-type hypersensitivity reactions(Brown L F et al J Immunol 154:2801, 1995).

Lymphangiogenesis mediated by VEGF-C and VEGF-D results from theirbinding to the FLT4 receptor or VEGFR3, expressed in the lymphaticendothelium. In some cases, even when over-expression of the ligands isnot present, the over-expression of the receptor has been related to anadverse prognosis in the course of a group of pathologic entities,including: diabetic retinopathy (Smith G. Br J Opthalmol 1999 April;83(4):486-94), chronic inflammation and ulcers (Paavonen K et al, Am JPathol 156:1499, 2000), the establishment of metastasis in lymphaticnodes and progression of breast cancer (Gunningham S P. Clin Cancer Res6:4278, 2000 Valtola R et al. Am J Pathol 154:1381, 1999), associated tonasopharyngeal tumors and squamous oral carcinomas (Saaristo A et al. AmJ Pathol 157:7, 2000; Moriyama M et al. Oral Oncol 33:369, 1997).Moreover, the over-expression of VEGFR3 is a sensitive marker of Kaposisarcoma, type Dabska hemangioendothelioma and of cutaneouslymphangiomatosis (Folpe A L et al. Mod Pathol 13:180, 2000; LymboussakiA et al. Am J Pathol 153:395, 1998).

Recently, two receptors where identified for VEGF named NRP1 and NRP2.These belong to the neurophilins family (NRP), and act as co-receptorsfor some specific isoforms of proteins of the VEGF family: VEGF-A₁₄₅VEGF-A₁₆₅, VEGF-B₁₆₇ and PIGF1, increasing their mitogenic capacity. Theexpression of NRP1 has become a marker of the aggressiveness of prostatecancer, has been related to the increase of angiogenesis in melanomas,and with apoptosis escape events in breast cancer (Latil A et al. Int JCancer 89:167, 2000; Lacal P M J Invest Dermatol 115:1000, 2000;Bachelder RE Cancer Res 61:5736, 2001). The coordinate over-expressionof NRP1, KDR, and VEGF-A₁₆₅ have been related to the fibrovascularproliferation in diabetic retinopathy cases and rheumatoid arthritis(Ishida S. et al. Invest Opthalmol Vis Sci 41: 1649, 2000; Ikeda M. Etal. J Pathol 191:426, 2000). NRP2 is over-expressed in osteosarcomaswhere it promotes angiogenesis and tumor growth (Handa A et al. Int JOncol 17:291, 2000).

Most of the therapeutic strategies based on angiogenesis inhibition,especially in cancer treatment, are based in the blockade of moleculesof the VEGF family and their receptors, with clinical trials in courseusing: (1) monoclonal antibodies blocking VEGF or the KDR receptor, (2)metalloproteinase inhibitors, as Neovastat and Prinomastat, (3) VEGFinhibitors as Thalidomide, Suramin, Troponin I, and IFN-α and Neovastat,(4) blockers of VEGF receptors as SU5416, FTK787 and SU6668, (5)inducers of tumor endothelium apoptosis, as Endostatin and CA4-P, and(6) ribozymes that decrease VEGF or VEGF receptors expression(Angiozyme). Due to the high homology between human VEGF and itsreceptors KDR and Flt-1 with their murine homologs (˜90%, 81%, and 89%,respectively), many animal models are used routinely to evaluate thepreclinical effectiveness of antiangiogenic compounds directed to thissystem (Hicklin D J et al. DDT 6:517, 2001).

Passive administration of antibodies to VEGF or VEGFRs is successfullytested in different clinical phases in humans (Hicklin D J et al. DDT6:517, 2001). The anti-VEGF humanized monoclonal antibody A.4.6.1(Genentech, San Francisco, United States) is in phase III clinical trialfor the treatment of colon, breast, kidney, and lung tumors (Kim, K J etal. Nature 362:841, 1993; Boersig C. R&D Directions October 7:44, 2001).In particular, for the case of the KDR receptor, a monoclonal antibodyhas been developed (IMC-1C11, ImClone) that recognizes the N-terminalextracellular domain of this receptor, and inhibits proliferation andmigration of leukemic human cells, increasing survival ofxenotransplanted mice. At present, its effect is being studied inpatients with colon cancer metastasis (Dias S et al. J Clin Invest106:511, 2000). In the aforementioned trials, the absence of concomitantadverse effects with the application of these monoclonal antibodies hasbeen demonstrated.

Notwithstanding the previous, a therapeutic modality not yet employedfor the blockade of neoangionegesis is specific active immunotherapy(SAI). In the SAI of cancer, antigens as peptides, proteins or DNA areemployed, mixed with appropriate adjuvants. SAI procedures pursue thestimulation of an immune response, both of the humoral (activation ofB-lymphocytes), and cellular types (activation of T helper, andcytotoxic lymphocytes, and natural killer cells), associated todendritic cell function as professional presenting cells in the MCHI andMHC II contexts (Bystryn J C, Medscape Hematology-Oncology 4:1, 2001;Parker, K C et al., J. Immunol 152:163, 1994; Nestle F O et al., NatureMedicine 7:761, 2001; Timmerman J M, Annual Review Medicine 50:507,1999).

SAI is a rapidly growing field of experimental and clinical research,with attractive applications, especially in oncology, where more than 60undergoing clinical trials based in procedures of SAI are reported,which surpass at present the clinical trials based on the use ofmonoclonal antibodies. In the particular case of cancer, the antigensused as immunogens for SAI are selected because of their physiologicalrelevance and difficulty of being substituted in the processes of tumorphenotypic drift (Bodey B et al., Anticancer Research 20: 2665, 2000),and because of their high specific association with the growth andevolution of tumor tissues.

The strategy of treating cancer using SAI also considers preferably theidentification of antigens expressed in different tumor types, whatcould increase the number of indications for the same vaccinepreparation. Examples of these are carcinoembryonic antigen (CEA),HER2-neu, human telomerase, and gangliosides (Greener M., Mol Med Today6:257 2000; Rice J, et al. J Immunol 167:1558, 2001; Carr A et al,Melanoma Res 11:219, 2001; Murray J L, et al. Semin Oncol 27:71, 2000).

In human tumors, VEGF is over-express in the tumor compartment (Ferrara,N. Curr. Top. Microbiol. Immunol. 237:1, 1999), and high levels of VEGFand its receptors have been demonstrated in the tumor-associatedvasculature (Brekken R A. J Control Release 74:173, 2001). The stromalcells also produce VEGF in response to the stimulus of transformedcells, with the result that when tumor cells are removed, VEGF levelspersist in the patients. The presence of VEGF and its receptors have apractical value for the establishment of prognosis and staging in casesof prostate, cervix, and breast tumors (George D J et al. Clin CancerRes 7:1932, 2001; Dobbs S P et al. Br J Cancer 76:1410, 1997; Callagy Get al. Appl Immunohistochem Mol Morphol 8:104, 2000). On the other hand,VEGF is also within the group of soluble factors that, together withother cytokines like IL-10, TNF-α and TGF-β, (Ohm J E y Carbone D P,Immunol Res 23:263, 2001), could be implicated in the immunosuppressionthat characterizes cancer patients (Staveley K, et al. Proc Natl AcadSci USA 95:1178, 1998; Lee K H, et al. J Immunol 161:4183, 1998). Thisimmunosuppressive effect seems to be related to its binding to the Flt1receptor (Gabrilovich D et al. Blood 92:4150, 1998).

The present invention describes procedures of SAI in experimental tumorsusing molecules of the VEGF family and their receptors. The antitumoraleffects obtained could be based in at least four different mechanisms,without discarding their possible combinations: (a) direct destructionof cancer and stromal cells producing VEGF, by cytotoxic lymphocytes,(b) damaging of endothelial cells of tumor-associated vessels due to thecapture or neutralization of the circulating VEGF via antibodies, (c)direct destruction of endothelial cells that express VEGF receptors, bycytotoxic lymphocytes or complement fixing antibodies, (d) activation ofa local immune response as a consequence of the capture orneutralization of circulating VEGF, and its consequent elimination ofits immunosuppressive effects.

Ideally, these treatments could be used to diminish or avoid theappearance of metastasis, to reduce or eliminate primary tumors as afirst or second line therapy, in combination or not with otheranti-tumor agents.

Active immunization directed to VEGF family and their receptors couldalso be efficient in the single or combined therapy of acute and chronicinflammatory processes (asthma, respiratory distress, endometriosis,atherosclerosis, tissular edema), infectious diseases (Hepatitis, Kaposisarcoma), autoimmune diseases (diabetes, psoriasis, rheumatoidarthritis, thyroiditis, synovitis), diabetic and newborn retinopathies,organ transplant rejection, macular degeneration, neovascular glaucoma,hemangioma, and angiofibroma, among others.

DETAIL DESCRIPTION OF THE INVENTION

According to the present invention, the in vivo administration ofoligonucleotide sequences encoding for proteins of the VEGF family,their receptors, co-receptors or their fragments, as well as of theirpolypeptidic variants, induces a cellular and humoral immune responsewith antiangiogenic and antitumoral effect.

Immunogens of polypeptidic nature of interest for the present invention,as well as their fragments, can be isolated from their natural sourcesor obtained by synthesis or recombinant DNA technology. Thesepolypeptides can also be produced fused to proteins with acknowledgedadjuvant activity like p64K (R. Silva et al U.S. Pat. No. 5,286,484 y EP0474313), or can be covalently bound to them after their individualobtainment. Other available strategy in these cases is the obtainment ofthe natural polypeptide, its mutated or modified variants, and theirfragments, as a part of loops exposed or not in bacterial proteins likeOMP1, which are part of immunostimulatory preparations, in thisparticular case VSSP (R. Perez et al U.S. Pat. No. 5,788,985 y 6149921).Furthermore, it is possible to obtain the polypeptidic immunogen exposedin the surface of a viral particle (HbsAg, VP2 of parvovirus, etc.),bound to specific peptides that target cells or organs specialized inthe induction of the immune response (CTLA4, Fc segment of the Ig,etc.), or to proteins capable of increasing biodistribution like VP22.

The principal natural sources of the proteins of interest for thepresent invention are predominantly expressed in placenta, activatedendothelial cells, and tumor cells. The mRNA of these cells or tissuesis used to obtain complementary DNA (cDNA) by known methods. Theextracted RNA is used as template for the amplification through thepolymerase chain reaction (PCR) of the cDNA corresponding to theselected antigen. In each case, primers used are designed according tothe characteristics of the vector where the cDNA is going to be insertedand to the previously reported sequences of the protein of interest.Alternatively, and preferably in the case of the receptors amplified byPCR, that are the largest size antigens that are used in the presentinvention, the coding regions are amplified in two or more overlappingfragments. These fragments include a common ligation site used toassemble the intact DNA, starting with its fragments.

An alternative for the cloning of the antigens of interest is theselection from commercially available DNA libraries derived from humanendothelium, or from tumors of this same origin. In some cases, it mightbe desirable to mutate some of the antigens object of the presentinvention, in order to avoid, especially with the VEGF family, anangiogenesis induction event produced by vaccination. These mutationsare made preferably in the receptor binding sites already described inthe literature. For this, primers are designed that cover both ends ofthe desired molecule, and the PCR products are used as template toobtain the mutated molecule. These mutated variants lack biologicalactivity but reproduce the immunogenic properties of the selectedantigen.

The cDNA molecules obtained by the methods described earlier areadministered in a proper vector, being this a virus, a plasmid, abacterial artificial chromosome, or similar. The vector carries theelements needed for the adequate expression of the gene in target cells,as well as the rest of elements that allows it to be produced in thehost cellular system according to its nature. DNA molecules of thepresent invention might contain one or more genes of interest,constituted by one or more nucleic acids (cDNA, gDNA, synthetic orsemi-synthetic DNA, or similar) that when transcribed or translated(when appropriate) in target cells generates the products withtherapeutic/vaccine value.

Generally, the gene of the vaccine therapeutic product according to theinvention is under the control of a transcriptional promoter that isfunctional in the target cell or the organism (mammals), as well as of a3′ end region that contains the signals needed for termination andpolyadenilation of the mRNA of the product of interest, allowing itsexpression. The promoter can be the natural promoter of the gene or aheterologous promoter transcriptionally active in the target cell. Thepromoter can be from eukaryotic or viral origin. Among eukaryoticpromoters, it is possible to use any promoter or derived sequence thatstimulates or represses the gene transcription, specifically or not,inducible or not, in a strong or weak manner. Additionally, the promoterregion can be modified by the insertion of activators or inductorsequences, allowing the tissue-specific or predominant expression of thegene in question.

Besides, the gene of interest can contain a signal sequence forsubcellular localization, in a way that its cellular localization orsecretion could be modified in the cell where it is expressed, orelsewhere, once synthesized. It can also contain a sequence encoding fora region of specific binding to a ligand specific of immune tissue,being directed to the site where the response is generated, with theobtainment of the therapeutic/vaccine effect.

Additionally, the gene of interest can be preceded by the codingsequence for the mRNA replication machinery, in a way that mRNA isamplified in the target cell, increasing the expression of said gene,and with it, of the therapeutic/vaccine effect according to theinvention. The replication machinery in question could of alphavirusorigin (Schlesinger S., Expert Opin Biol Ther. 1:177, 2001), morespecifically derived from the Sindbis or Semliki viruses, or similar. Inthis particular case, the gene of interest is under the transcriptionalcontrol of a subgenomic promoter that allows the amplification of itsmRNA in target cells, once the molecules according to the presentinvention have been internalized. Besides, the DNA vector might containsequences that permit the replication of the molecules object of thepresent invention in mammalian cells. This allows an increase in theexpression levels and/or of the therapeutic/vaccine effect (Collings A.,Vaccine 18: 4601, 1999)

The DNA vector can be purified using standard techniques for plasmid DNApurification. These techniques include the method of purification bycesium chloride density gradient, in the presence of ethidium bromide,or alternatively, the use of ionic exchange columns or any otherexchanger or method to separate DNA molecules (Ferreira G N, et al,Trends Biotechnol. 18:380, 2000).

The present invention includes the use of plasmidic DNA vectors,preferably those of the PAEC family of compact vectors for DNAimmunization and gene therapy in humans (Herrera et al, Biochem.Biophys. Res. Commu. 279: 548, 2000). This family comprises vectorspAEC-K6 (Access number AJ278712), pAEC-M7 (Access number AJ278713),pAEC-Δ2 (Access number AJ278714), pAEC-SPE (Access number AJ278715) andpAEC-SPT (Access number AJ278716). These vectors contain only theessential elements for the expression of the product of interest inmammalian cells, including human cells, and a replication unit inEscherichia coli. The transcriptional unit is formed by the immediateearly promoter of human cytomegalovirus (CMV), a versatile multicloningsite for the insertion of the product of interest, and the sequences fortranscriptional termination and polyadenilation derived from simianvirus 40 (SV40). In the replication unit, the vector contains the genefor kanamycin resistance (Tn903), and a pUC19 replication origin(ColE1), in order to guarantee a high copy number and the selection ofthe bacteria that bear the plasmid of interest.

Furthermore, the present invention includes the use of plasmidic DNAvectors, preferably those of the PMAE family of compact vectors for DNAimmunization in humans. These contain the same functional elements inbacteria as PAEC series, as well as the CMV immediate early promoter andthe multicloning site. Additionally, they bear a synthetic intron and asynthetic sequence for transcription termination and polyadenilation,derived from rabbit β-globin. It has been reported that with sequencessimilar to the latter it is possible to obtain higher expression levelsof the cloned gene (Norman J A et al, Vaccine 15: 801, 1997). Moreover,the vectors of this series include consecutive repetitions ofimmunostimulatory sequences (CpG motives), that stimulate innate immunesystem in both mice and humans, with the consequent activation of ahumoral and cellular response against the molecule of interest (Krieg AM, Vaccine 19:618, 2001).

The immunization with recombinant virus (adenovirus, adeno-associated,vaccinia, chickenpox virus, canarypox virus, among others) produces apotent cytotoxic cellular response in the hosts. To introduce thesequence of interest in the recombinant virus vectors that haveintegration sequences and promoters that are particular for each virustype, are used. This strategy is also included in the scope of thepresent invention, and chickenpox virus and the pFP67xgpt vector arepreferably used. The pFP67xgpt vector is used to clone genes under astrong early/late promoter of synthetic nature between the open readingframes 6 and 7 of a fragment of 11.2 kB BamHI of the chickenpox virusFP9. This plasmid also contains the Ecogpt controlled by the vacciniapromoter p7.5K, which is used to identify recombinant virus.

Other alternative of the present invention consists of the immunizationwith proteins of the VEGF family and their receptors and/orco-receptors. cDNA molecules obtained as previously described are clonedin vectors for expression in virus, yeast, phage, plants, or superiorcells, in order to obtain the protein variants of the antigens, aftertheir sequence has been verified by the traditional methods of automaticsequencing. Several vectors for expression have been described and usedfor the obtainment of recombinant proteins. These vectors contain, atleast, a sequence that controls the expression operatively linked to thesequence of the DNA or fragment to be expressed. Examples of sequencesuseful for the control of expression are: the systems lac, trp, tac, andtrc, the promoter regions and the principal operator of lambda phage,the controller region of the surface protein fd, the glycolyticpromoters of yeast (for example, the 3-phosphoglicerate kinase), thepromoters of yeast acid phosphatase (for example, Pho5), the yeastpromoters for the mating alpha factor, and the promoters derived frompolyoma, adenovirus, retrovirus, simian virus (for example, theearly/late promoters of SV40), and other known sequences that regulatethe expression of genes in prokaryotic and eukaryotic cells, theirviruses, and their combinations.

The hosts used for the replication of these vectors and the obtainmentof the recombinant proteins object of the present invention includeprokaryotic and eukaryotic cells. The prokaryotic comprise E. coli (DHI,MRCI, HB101, W3110, SG-936, X1776, X2282, DH5a), Pseudomonas, Bacillussubtilis, Streptomices, and others. The eukaryotic cells include yeastand fungi, insects, animal cells (for example, COS-7 and CHO), human,and plant cells, and tissue cultures, among others. After the expressionin the system of choice in an adequate media, the polypeptides orpeptides can be isolated by known procedures.

Use of Adjuvants

Even when vaccination with naked DNA or proteins has shown to beeffective in certain animal models, the patients affected by tumors orautoimmune diseases present a challenge to the therapeutic strategyproposed by the present invention. To favor the immune response, the DNAor protein vaccines can be combined with immunopotentiators alreadydescribed like: mineral salts (ex., Aluminum hydroxide, aluminumphosphate, calcium phosphate); immunostimulators like: cytokines (ex.,IL-2, IL-12, GM-CSF, IFN-α, IFN-γ, IL-18), molecules (ex., CD40, CD154,invariant chain of MHC type I, LFA3); saponins (ex., QS21), MDPderivatives, CpG oligos, LPS, MPL and polyphosphazenes; lipidicparticles: like: emulsions (ex., Freund, SAF, MF59), liposomes,virosomes, iscoms, co-chelators; microparticular adjuvants like PLGmicroparticles, poloxamers, of viral type (ex., HBcAg, HCcAg, HBsAg),and of bacterial type (ie., VSSP, OPC); and mucosal adjuvants likeheat-labile enterotoxin (LT), cholera toxin, and mutant toxins (ex.,LTK63 y LTR72), microparticles and polymerized liposomes. In the case ofDNA vaccination, the expression of the antigen of interest could becombined with some of the immunopotentiator molecules already mentioned,on a bi-cistronic vector.

The experimental situations detailed in the examples demonstrate thatDNA can be coupled in a non-covalent manner to some of the mentionedparticles and that the use of these mixtures reduce the optimalconcentration to obtain an anti-tumor response, similar to thosedescribed for higher doses of naked DNA.

Administration to a Mammal

For the therapeutic applications, the vaccine preparations of thepresent invention are administered to a mammal, preferably a human, in adose pharmaceutically acceptable, by the following routes: mucosal,subcutaneous, intramuscular, peritoneal, intra-lymphatic, topic, and byinhalation, among others. These could be administered on the tissueinterstitial space, including: muscle, skin, brain, lung, liver, bonemarrow, spleen, thymus, heart, lymph nodes, blood, bone, cartilage,pancreas, kidney, bladder, stomach, intestine, testicles, ovary, uterus,rectum, eye, glands, and connective tissue. In the case of vectors foroligonucleotide transfer, their expression is preferably directed tosomatic differentiated cells, though they may be directed tonon-differentiated or less differentiated cells like skin fibroblastsand blood pluripotent cells.

The doses of the immunogen could be administered in pharmaceuticallyaccepted vehicles without toxicity or therapeutic effects. Examples ofthese vehicles include: ionic exchangers, alumina, aluminum esthearates,lecitine, seric proteins like albumin, buffer solutions, likephosphates, glycine, sorbic acid, potassium sorbate, partial glyceridemixtures of saturated fatty acids of plant origin, water, salts, orelectrolites, like protamine sulphate, di-sodic hydrogen phosphate,sodium chloride, zinc salts, colloidal silica, magnesium trisilicate,polyvinyl pirrolidone, substances base on cellulose and polyethyleneglycol. In the present invention, preferably phosphate buffers asvehicles of the vaccine preparations are used.

In the case of the use of proteins and peptides, these can be conjugatedin covalent or non-covalent manner to molecules known as carriers thatact like adjuvants. Among these molecules are: KLH, p64K, OPC (MusacchioA et al, Vaccine 19; 3692, 2001), and VSSP. The combination of nakedDNA, viral vectors, and protein immunogens is an alternative alsoincluded within the scope of the present invention. In an advantageousmanner, plasmid DNA administration allows the generation of formulationswith one or more molecules of interest in the vaccine preparation. Thus,molecules according to the present invention can be administered invaccine schedules through the combination of different types of vectors(variant of induction re-stimulation, with DNA, proteins, viral vectors)

DNA vectors could be directly administered to the patient, or host cellscan be in vivo or ex vivo modified with these vectors. This laststrategy can be combined with the insertion by site-specificrecombination or the immunization by somatic transgenesis that directsthe vector expression to specific cells. Furthermore, bacterial hosts ofDNA vectors could be used as their vehicles of transfer in vivo.

In this way, the molecules carrying the genes according to the presentinvention could be used in the form of naked DNA, or in combination withdifferent vectors: chemical/biochemical/biologic, natural/synthetic orrecombinant. These molecules can be coupled or combined with cationicpeptides, compacting molecules (ex. PEG, PEI), nuclear localizationpeptides (NLP), etc. These could be administered also together withcations capable of forming DNA precipitates, as a part of liposomalpreparations to which the molecules have been added previously to themembrane fusion, and in synthetic vectors of lipid nature, or formed bycationic polymers (ex. DOGS or DOTMA). For the administration of the DNAvectors, chimeric proteins able to compact DNA and mediate the transportof the complex formed, and its selective endocytosis by specific cells,can also be used. DNA molecules carrying the therapeutic/vaccine genesaccording to the invention could be used for the genetic transfer tocells using physical methods of transfer, like particles bombardment,electroporation (in vitro, in vivo or ex vivo), or directly in vivo bytopic application, inhalation by particulation, etc. The live vectorsinclude adenoviral particles or the same hosts where the moleculesaccording to the present invention have been generated.

The doses of polypeptides and/or oligonucleotides to be used can beestablished according to different parameters, in particular dependingon the gene or protein administered as an immunogen, the route ofadministration, the pathology to be treated, the period of treatment,and in the case of using oligonucleotides, of the vector used forimmunization. A change in dose schedule or administration routedifferent to those described in the following examples, do not separatefrom the principle or precept of the present invention, being possibleto achieve an optimization of the immunization schemes to obtain abetter response.

Therapeutic Uses

The present invention has advantages over passive immunotherapy, whichis in advanced phases of clinical trials using the same molecules astargets. In comparison with passive transfer of immunity through theadministration of monoclonal antibodies (ex. Anti-VEGF), theimmunization, be it with the protein or the oligonucleotide, has theadvantage of inducing the endogen production of antibodies and inaddition the proliferation and expansion of specific cytotoxic CD8+lymphocytes.

The present invention has advantages over the therapeutic strategiesdirected to block VEGF-VEGFRs system, mainly because these strategiesonly diminish the levels of circulating VEGF or block KDR. The strategyproposed, apart from achieving the mentioned effects, also destroys thesource of VEGF (that is, the tumor cells and associated stroma) and/orthe cells expressing their receptors (tumor endothelium and some tumorcells). Previous work done in this area only describe a humoral responseas a principal component of the observed effect. Without the intentionof limiting the scope of the present invention to a particularmechanism, the examples show that, besides from the humoral specificresponse, the vaccine compositions are able to elicit a CD8+ cellularresponse that cooperates with the humoral response; and that in thetumor context, the combination of both are relevant to obtain anant-tumor effect, the previous observed in example 9.

It is possible that the cytotoxic cellular response is mediated by therecognition of some of the peptides that appear in Tables 1 and 2. Inthese, some peptidic segments appear, that could be relevant in thecellular response directed to selected targets in VEGF family, itsreceptors and co-receptors. This information was obtained throughcomputer analyses on public databases from NIH and Heidelberg Institute(http://bimas.dcrt.nih.gov/molbio/hla_bind, andwww.bmi-heidelberg.com/scripts/MHCServer.dll/home.htm) using BIMAS andSYFPHEITI software, respectively. The peptides marked and othersequences derived from the antigens of interest could be used for theactive immunotherapy of the already described pathologies, as a singleor combined treatment, and as part or not of molecules with adjuvantcapacities. These peptides can also be used in their oligonucleotidevariants with vaccine purposes.

The methods to inhibit angiogenesis and the pathologic conditionsassociated to this event, comprise the administration of an effectiveamount of the DNA or protein of some of the molecules described in thisinvention, by any of the routes, and with the use of some of thepreviously described immunopotentiators or adjuvants, to a mammal. Thismammal is preferably a human.

A non-reversible and unregulated increase of angiogenesis has beenrelated to a wide group of diseases. The system that comprises the VEGFfamily, its receptors and co-receptors is over-expressed in many ofthese pathological conditions, as it has been described before. In thisway, the therapeutic strategies proposed by the present invention resulteffective in the treatment of: (a) cancer (both primary tumors and theirmetastasis), (b) acute and chronic inflammatory processes like asthma,respiratory distress, endometriosis, atherosclerosis, and tissularedema, (c) diseases of infectious origin like Hepatitis and Kaposisarcoma, (d) autoimmune diseases like diabetes, psoriasis, rheumatoidarthritis, thyroiditis, and (e) other diseases and states such asdiabetic and newborn retinopathies, organ transplant rejection, maculardegeneration, neovascular glaucoma, hemangioma and angiofibroma.

Particularly in the case of cancer, vaccination with the immunogensproposed by the present invention results effective in the treatment ofcarcinomas, sarcomas and vascularized tumors. Some examples of tumorsthat can be treated with the proposed strategies include epidermoidtumors, squamous tumors like those of the head and neck, and colorectal,prostate, breast, lung (including small and non-small cells), pancreas,thyroid, ovary, and liver tumors. These methods are also effective inthe treatment of other types of tumors, like Kaposi sarcoma, centralnervous system neoplasia (neuroblastoma, capillary hemangioma,meningioma and brain metastasis), melanomas, renal and gastrointestinalcarcinomas, rhabdomyosarcoma, glioblastoma and leiomiosarcoma.

Specifically the use of VEGF-A and/or their receptors VEGFR-1 andVEGFR-2 as immunogen is useful for the treatment of: tumors of differentorigins and localizations and their metastasis, of hemangioma, ofendometriosis, of tissue edemas, of chronic inflammatory processes likeulcerative colitis and Crohn's disease, of, atherosclerosis, ofrheumatoid arthritis and osteoarthritis, of inflammatory arthropathies,psoriasis, respiratory distress, asthma, thyroidits, of diabetic andnewborn retinopathies, macular degeneration, and glaucoma, of theautosomic VHL disease, of obesity, and of the rejection of some organtransplants. On the other hand, a response vs PIGF is useful in cases ofrheumatoid arthritis and in general for the treatment of primaryinflammatory arthropathies.

In the case of VEGF-B, its use as immunogen results useful in cases ofbreast, ovary, and kidney tumors, and for melanoma and fibrosarcoma. Theuse of VEGF-C and its receptor VEGFR-3 results useful in the treatmentof tissular edema, diabetic retinopathy, chronic inflammation, ulcers,and tumors of the breast, lung, head and neck, esophagus, stomach,lymphomas, and prostate, metastatic nodules and Kaposi sarcoma, Dabskatype hemangioendothelioma and of the cutaneous lymphangiomatosis.Immunization with VEGF-D can be used specifically for the treatment oflymphatic node metastasis.

The use of NRP1 and NRP2 co-receptors in mammal immunization resultsuseful for the treatment, in particular, of fibrovascular proliferationin prostate cancer, melanoma, osteosarcoma, breast cancer metastasis,diabetic retinopathy, and rheumatoid arthritis.

The studies based on the passive immunotherapy by administration ofantibodies have shown that the combination of antibodies vs VEGF-A andKDR is more effective in models of syngeneic tumors. Thus, the use oftwo or more of the immunogens proposed in the present invention providesan especially efficient treatment for the inhibition of angiogenesis andtumor growth. These immunogens can be administered in an individualmanner or by pairs using bi-cistronic vectors by the already mentionedpathways. Furthermore, vaccine compositions of the invention can be usedtogether with, or in sequential manner, with drugs or chemotherapeuticagents, that offer a benefit to the condition under treatment.

The results described below demonstrate that the anti-angiogenic andanti-tumor responses are mediated by a cooperation of the humoral andcellular responses. In particular, VEGF and its receptor are involved inthe process of maturation of dendritic cells and act on B and Tlymphocytes precursors. Example 10 demonstrates that the proposedtherapeutic strategy, apart from diminishing the levels of VEGF in seraalso contributes to the normalization of the proportions of B and Tlymphocytes, and of mature dendritic cells. This effect favors thepresentation of tumor antigens within the MHC I context, improving thequality and intensity of the immune anti-tumor response directed notonly to the immunogen, but also to the other tumor-associated,tumor-specific, and over-expressed antigens, in the tumor context.

TABLE 1 Estimation of the VEGF protein family MHCI associated peptidesin the context of HLAA.0201 A.-Using BIMAS software VEGF-A VEGF-B VEGF-CVEGF-D PIGF Sequence Kd Sequence Kd Sequence Kd Sequence Kd Sequence KdLLSWVHWSL 272 LLLAALLQL 309 YLSKTLFEI 640 FMMLYVQLV 1966 RLFPCFLQL 150ALLLYLHHA 42 QLAPAQAPV 70 TLFEITVPL 324 KLWRCRLRL 620 VVSEYPSEV 42WSLALLLYL 30 QLVPSCVTV 70 VLYPEYWKM 304 QLFEISVPL 324 VMRLFPCFL 42FLQHNKCEC 23 LMGTVAKQL 26 CMNTSTSYL 85 YISKQLFEI 88 RALERLVDV 34WVHWSLALL 20 LLAALLQLA 19 KLFPSQCGA 64 CMNTSTSYI 41 VELTFSQHV 32FLLSWVHWS 16 LLQLAPAQA 8 LLGFFSVAC 32 VLQEENPLA 35 AVPPQQWAL 14RQLELNERT 6 WSWIDVYT 6 SLPATLPQC 11 WVVVNVFMM 27 LQLLAGLAL 14 NITMQIMRI3 CVPTGQHQV 6 GLQCMNTST 7 VNVFMMLYV 10 RSGDRPSYV 10 YCHPIETLV 2KQLVPSCVT 4 AAFESGLDL 4 SLICMNTST 7 LLAGLALPA 8 IEYIFKPSC 2 VVVPLTVEL 3EQLRSVSSV 4 CVLQEENPL 7 CVPVETANV 6 B.-Using SYFPEITHI software VEGF-AVEGF-B VEGF-C VEGF-D PIGF Sequence Score Sequence Score Sequence ScoreSequence Score Sequence Score LLSWVHWSL 24 LLLAALLQL 29 TLFEITVPL 27FMMLYVQLV 25 ALERLVDVV 26 ALLLYLHHA 24 QLAPAQAPV 26 DLEEQLRSV 26QLFEISVPL 25 RLFPCFLQL 24 WVHWSLALL 20 QLVPSCVTV 26 YLSKTLFEI 26YISKQLFEI 24 RALERLVDV 24 SLALLLYLH 20 VVVPLTVEL 24 ALLPGPREA 24KLWRCRLRL 23 LLAGLALPA 22 SYCHPIETL 19 LLRRLLLAA 23 CMNTSTSYL 21RAASSLEEL 22 LAGLALPAV 22 NITMQIMRI 19 LLAALLQLA 23 DICGPNKEL 21SLEELLRIT 22 VMRLFPCFL 20 FLLSWVHWS 18 FLRCQGRGL 22 AAAAFESGL 20ATFYDIETL 22 CFLQLLAGL 20 WSLALLLYL 18 LTVELMGTV 21 AAFESGLDL 20EISVPLTSV 22 QLLAGLALP 20 HPIETLVDI 18 LRRLLLAAL 20 VLYPEYWKM 20SLICMNTST 20 SAGNGSSEV 20 CNDEGLECV 18 LMGTVAKQL 19 IIRRSLPAT 20VPLTSVPEL 20 VVSEYPSEV 20 Note: Values in bold correspond to thosepeptides or their regions, which coincide in both predictions.

TABLE 2 Estimation of VEGF family receptors MHCI associated peptides inthe context of HLAA.0201 A.-Using BIMAS software VEGFR-1 VEGFR-2 VEGFR-3NRP-1 NRP-2 Sequence Kd Sequence Kd Sequence Kd Sequence Kd Sequence KdFLYRDVTWI 1942 VLLWEIFSL 1792 VLLWEIFSL 1793 GLLRFVTAV 2249 WMYDHAKWL5121 VLLWEIFSL 1792 SLQDQGDYV 769 RLLEEKSGV 1055 VLLGAVCGV 1006ILQFLIFDL 484 KLLRGHTLV 901 VLLAVALWL 739 VLWPDGQEV 981 WMPENIRLV 436YLQVDLRFL 247 GLLTCEATV 257 AMFFWLLLV 427 NLTDLLVNV 656 GILSMVFYT 278ALYFSRHQV 223 TLFWLLLTL 182 VIAMFFWLL 270 KQAERGKWV 557 LLCAVLALV 272NMLGMLSGL 131 ILLSENNVV 179 ILLSEKNW 179 GVIAVFFWV 369 VLLHKSLKL 134WLYTLDPIL 129 TLNLTIMNV 160 LLAVALWLC 146 KLVIQNANV 243 GMLGMVSGL 131DIWDGIPHV 56 CVAATLFWL 137 KNLDTLWKL 128 ALWNSAAGL 177 FQLTGGTTV 120KMEIILQFL 44 LLSIKQSNV 118 AVIAMFFWL 113 TLSLSIPRV 160 VLATEKPTV 118VLNKLHAPL 36 SLQDSGTYA 112 LLLVIILRT 108 SQHDLGSYV 159 GPFLFIKFV 81LLGATCAGL 36 B.-Using SYFPEITHI software VEGFR-1 VEGFR-2 VEGFR-3 NRP-1NRP-2 Sequence Score Sequence Score Sequence Score Sequence ScoreSequence Score TLFWLLLTL 29 VLLWEIFSL 29 VLLWEIFSL 29 VLLGAVCGV 30NMLGMLSGL 27 VLLWEIFSL 29 LLVIILRTV 28 SIPGLNVTL 27 GLLRFVTAV 29ILQFLIFDL 26 ILGPGSSTL 28 GLFCKTLTI 26 NLTDLLVNV 27 LLCAVLALV 28DIWDGIPHV 26 LLCALLSCL 27 SIMYIVVVV 26 VLWPDGQEV 26 GMLGMVSGL 28YLQVDLRFL 26 GLLTCEATV 27 IILVGTAVI 26 LLPRKSLEL 26 ALGVLLGAV 28TLDPILITI 26 LLRGHTLVL 27 ALMSELKIL 26 ALWNSAAGL 26 VLLHKSLKL 27ILAKPKMEI 25 ALMTELKIL 26 AASVGLPSV 25 IMDPGEVPL 26 VLATEKPTV 26VLNKLHAPL 25 KLLRGHTLV 25 SISNLNVSL 25 RLWLCLGLL 25 QLTGGTTVL 25LLGATCAGL 25 TLNLTIMNV 25 AMFFWLLLV 25 LIYFYVTTI 25 VLLGAVCGV 30ALYFSRHQV 23 ILLSENNVV 25 ILLSEKNW 25 LLEGQPVLL 25 GLLRFVTAV 29GIGMRLEVL 23 Note: Values in bold correspond to those peptides orregions, which coincide in both predictions.

EXAMPLES Example 1 Cloning and Transient Expression of Antigens HumanVEGF, its Isoforms and Functional Mutants

VEGF isoforms were cloned applying the polymerase chain reaction (PCR)using as template a cDNA obtained from a previous isolation of mRNA ofCaSki cell line (ATCC CRL 1550), according to the manufacturerinstructions (Perkin-Elmer), and utilizing primers SEQ ID1 and SEQ ID2.Bands corresponding to the amplification products of VEGF isoforms 121,165 and 189 were extracted from 2% agarose gels. After band digestionwith endonucleases BamHI and EcoRI, the cDNAs from the VEGF isoformswere purified and cloned independently in the PAECΔ2 vector (proprietaryvector of CIGB). Resulting plasmids were sequenced and determined tohave no mutations with respect to the aminoacid sequences reported bythe EMBL (www.embl-heidelberg.de) for the cloned isoforms. The cDNAcorresponding to VEGF isoforms were subsequently cloned KpnI/EcoRV onthe pMAE5Δ5 vector, that among other characteristics differs from pAECΔ2by the presence of 5 immunostimulatory CpG sites.

cDNA from a VEGF variant deficient for the binding to the KDR receptor(VEGF_(KDR(−))) was obtained by direct mutagenesis of the VEGF₁₂₁isoform previously cloned, as described by Siemeister G et al(Siemeister G et al. J Biol Chem 273:11115, 1998). The mutated variantwas generated by PCR using the following primers:

(A) Amplification of the 5′ terminal fragment (315 bp): using primerswith sequences SEQ ID3 and SEQ ID4

(B) Amplification of the 3′ terminal fragment (93 bp): using primerswith sequences SEQ ID5 and SEQ ID6.

The fragments thus amplified were purified as referred, and were used inequimolar concentrations as a template for a fusion PCR using theprimers corresponding to sequences SEQ ID7 and SEQ ID8. Resultant cDNAcontaining the mutation was digested BamHI/EcoRI, and was purified, andcloned in pAECΔ2 vector. The mutations introduced were checked bysequencing, and the DNA corresponding to VEGF_(KDR(−)) was subclonedKpnI/EcoRV in pMAE5Δ5 vector resulting in pMAE5Δ5 VEGF_(KDR(−)).

Plasmids used both in transfection and in animal vaccination werepurified in endotoxin-free conditions, as described by Whalen R. et al.(Whalen R G y Davis H L. Clin Immunol Immunopathol 75:1, 1995). Briefly,DNA was purified using QIAGEN Endo-free systems following themanufacturer instructions, and the DNA was furthermore submitted to asecond precipitation. Finally, DNA was dissolved in endotoxin-free PBS(SIGMA, USA) to a final concentration of 4 mg/mL.

1.2 Human VEGF Receptor (KDR/Flk1)

The cDNAs coding for the extracellular domain of KDR receptor of VEGF(KDR1-3) and for the transmembrane and intracellular domains of thisreceptor (KDR TC), were obtained from an RT-PCR using mRNA of theendothelial cell line HUVEC (Clonetic, USA), treated with human VEGF(Sigma) and Heparin (Sigma).

In the case of the extracellular domains 1 to 3, the primers usedcorrespond to sequences SEQ ID9 and SEQ ID10. After digestion of theamplified fragment (943 bp) with endonucleases BamHI and EcoRI, the cDNAcoding for 1-3 domains of KDR was purified, and cloned in pAECΔ2 vector.Clones positive by restriction analysis were verified by sequencing ofthe corresponding DNA. The cDNA corresponding to KDR 1-3 was thensubcloned KpnI/EcoRV in the already described pMAE5Δ5 vector (pMAE5Δ5KDR1-3).

For the cloning of transmembrane and cytosolic regions of the receptor atwo-step strategy was designed. For the insertion of the first segment,the primers corresponding to SEQ ID11 and SEQ ID12 were used. After theXbaI/BgIII digestion of this 747 bp segment, the product was cloned inthe pMAE5 vector, previously digested with the same enzymes, obtainingthe plasmid PMAE5 KDR 747. This plasmid was digested BglII/NotI in orderto insert the remaining carboxi-terminal fragment of 1091 bp that wasamplified using the primers corresponding to sequences SEQ ID13 and SEQID14. Clones positive by restriction analysis were verified by DNAsequencing and denominated pMAE5 KDR C.

1.2.1 Cloning of the transmembrane and cytosolic regions of KDR in aviral vector For the cloning of transmembrane and cytosolic regions ofVEGF receptor (KDR) on the chickenpox virus, the primers correspondingto sequences SEQ ID15 and SEQ ID16 were used. After digesting this 953bp segment with StuI/SmaI enzymes, the product was cloned in thepFP67xgpt vector, previously digested with the same enzymes. In thissame vector, digested SmaI/BamHI, the remaining 919 bp were inserted,that were amplified from the original cDNA using primers correspondingto sequences SEQ ID17 and SEQ ID18. Clones positive by restrictionanalysis were verified by DNA sequencing and denominated pFP67xgpt KDRC.

Chickenpox virus (FWPVs) were replicated in chicken embryo fibroblasts(CEF), in DMEM medium supplemented with 2% of fetal bovine serum (FBS).The pFP67xgpt KDR C was transfected using Lipofectin (Gibco BRL, GrandIsland, USA) in CEF previously infected with the attenuated strain FP9.After 24 hours, fresh medium was added and cells were cultured for other3 to 4 days. After this time, cells were frozen-thawed three times.Recombinant viruses expressing the gene coding for the Ecogpt enzymewere purified in selective media with mycophenolic acid (25 μg/mL),xantine (250 μg/mL), and hypoxantine (15 μg/mL) (MXH). The correctinclusion of the gene in recombinant viruses was checked by PCR.Recombinant viruses were denominated FPKDRgpt and non-recombinants usedas negative control FP.

Example 2 In Vivo Expression of Antigens

In order to confirm the potential of the constructions made to expressthe proteins in vivo, these were injected in the femoral quadricepsmuscle of C57BL6 mice (3 per group)

1. pMAE5Δ5-VEGF₁₂₁ (10 and 50 μg/mouse) in PBS pH 7.22. pMAE5Δ5-VEGF₁₆₅ (10 and 50 μg/mouse) in PBS pH 7.23. pMAE5Δ5-VEGF₁₈₉ (10 and 50 μg/mouse) in PBS pH 7.24. pMAE5Δ5-VEGF_(KDR(−)) (10 and 50 μg/mouse) in PBS pH 7.25. pMAE5Δ5-KDR 1-3 (10 and 50 μg/mouse) in PBS pH 7.26. pMAE5 KDR C (10 and 50 μg/mouse) in PBS pH 7.27. FPKDRgpt (2.5*10⁷ cfu) in PBS pH 7.28. PBS pH 7.2 (negative control)

48 hours after injection the animals were sacrificed and injectedmuscles extracted in one piece. A part of the muscular tissue washomogenized in presence of protease inhibitors and non-ionic detergents.Presence of VEGF in protein extracts was analyzed by Dot-Blot and byWestern-Blot using a polyclonal antibody that recognizes all human VEGFisoforms (sc-152G), following described procedures. RNA was extractedfrom the remaining muscular tissue using TRI-Reagent (SIGMA). A total of20 μg of RNA from each experimental situation were submitted toelectrophoresis in 1% agarose gels containing formaldehyde. RNA wastransferred to a nylon filter (HYBOND) and hybridized with the cDNA ofVEGF 121 isoform labeled with ATP³², that recognizes all VEGF isoforms,or with the cDNA of KDR similarly labeled. In both cases, filters werere-hybridized with the cDNA corresponding to a constitutive gene: thegliceraldehyde 3-phosphate dehydrogenase (GAPDH). In all the analyzedconstructions bands corresponding to human VEGF and the cloned fragmentsof the KDR receptor were identified.

Example 3 In Vivo Protection Experiments Employing Vaccination with thePlasmid Containing the Gene Fragments of KDR, the VEGF Receptor

Groups of 10 C57BL/6 mice were vaccinated or not with the followingvariants:

1. pMAE5Δ5-KDR 1-3 (1, 10, 50 and 100 μg/mouse) in PBS pH 7.22. pMAE5 KDR C (1, 10, 50 and 100 μg/mouse) in PBS pH 7.23. FPKDRgpt (2.5*10⁷ cfu)4. PBS pH 7.2 (negative control)5. FP (2.5*10⁷ cfu) (negative control group 3)

In every case, mice were immunized by intramuscular injection (im.) inthe rear left foot with a total volume of 50 μl. All the animals werere-immunized 15 days later using the original immunization regime. Thetumor challenge was developed thirty days after the last immunization,by a subcutaneous (sc.) injection of 10⁴ cells of B16-F10 melanoma(ATCC, CRL-6475) in the right ventral zone of every animal. Tumor growthwas monitored with three weekly measurements until animals started todie.

In mice immunized with the pMAE5Δ5-KDR 1-3 plasmid a reduction of tumorsize was evidenced at doses of 50 and 100 μg of DNA/mouse, significantlylower with respect to the negative control (Table 3). A survivalanalysis at day 33 revealed a significant increment (with respect to thenegative control) of this parameter for the animals immunized with thesaid DNA doses of 50 and 100 μg per mouse, with respect to theun-immunized mice (group PBS pH7.2). In the case of pMAE5Δ5-KDR C (Table3) a significant reduction of tumor volume was observed at the fourdoses used, with an increment in survival for doses from 100 to 10μg/animal. The use of viral vectors reduced the volume and increasedsurvival in the condition used for the FPKDRgpt construction (Table 3),in comparison to the respective negative control (group of miceimmunized with the vector without insert FPgpt).

TABLE 3 Tumor volume and survival in mice immunized with the fragmentsof the VEGF receptor (KDR) gene. [DNA Tumor Vol. (mm³) Survival Groupμg] Day 24 (Day 43) PMAE5Δ5- 100 424.0 ± 199.2 (***) (***) KDR1-3 50756.32 ± 435.9 (***) (**) 10 1024.2 ± 397.1 (*) (ns) 1 1334.2 ± 620.7(ns) (ns) pMAE5Δ5- 100 404.23 ± 200.0 (***) (***) KDR C 50 633.2 ± 365.2(***) (***) 10 924.3 ± 437.1 (**) (*) 1 1114.2 ± 665.7 (*) (ns) FPKDRgpt2.5*10⁷ cfu 304.23 ± 152.0 (***) (***) FPgpt 2.5*10⁷ cfu 1891.0 ± 726.0(ns) (ns) PBS pH 7.2 — 1785.0 ± 826.0 — Note Tumor volume is reported asmean ± standard deviation (SD) of the measures performed on the animalsof each group, statistical comparisons were performed using one-wayANOVA and a Bonferroni post-test. In the case of survival, the reportedstatistical significance was obtained using the log-rank test to compareeach group with respect to the control group, in the indicated day.Statistical signification is indicated as (ns) p ≦ 0.05 non-significant;(*) p ≦ 0.05; (**) p ≦ 0.01; and (***) p ≦ 0.001.

Example 4 In Vivo Protection Experiments Using Vaccination with thePlasmids Containing the VEGF Isoforms, and the Mutated Variant

Groups of 10 mice C57BL/6 were vaccinated or not with the followingvariants:

1. pAECΔ2-VEGF₁₂₁ (1, 10, 50 and 100 μg/mouse) in PBS pH 7.22. pMAE5Δ5-VEGF₁₂₁ (1, 10, 50 and 100 μg/mouse) in PBS pH 7.23. pMAE5Δ5-VEGF₁₆₅ (1, 10, 50 and 100 μg/mouse) in PBS pH 7.24. pMAE5Δ5-VEGF₁₈₉ (1, 10, 50 and 100 μg/mouse) in PBS pH 7.25. pMAE5Δ5 VEGF_(KDR(−)) (1, 10, 50 and 100 μg/mouse) in PBS pH 7.26. PBS pH 7.2 (negative control)

In every case, mice were immunized by im. injection in the rear leftfoot with a total volume of 50 μl. All the animals were re-immunized 15days later using the original immunization regime. The tumor challengewas developed thirty days after the last immunization, by a subcutaneousinjection of 10⁴ cells of B16-F10 melanoma (ATCC, CRL-6475) in the rightventral zone of every animal. Tumor growth was monitored with threeweekly measurements until animals started to die.

For the naked DNA variant in pAEC series in the case of mice immunizedwith 100 μg/animal, a decrease in tumor growth with respect to thenegative control was observed (Table 4). In the variants included in thevector of the pMAE5Δ5 series with 5 CpG sites, independently of the VEGFisoform, tumor size was significantly reduced as compared to thenegative control in the groups of mice immunized with doses of 10, 50,or 100 μg of DNA. In the case where the mutated variant pMAE5Δ5VEGF_(KDR(−)) was used, a significant reduction of tumor size wasobtained at similar doses as those employed with the pMAE5Δ5-VEGF₁₂₁.

A survival analysis on day 43 evidenced a significant increase (withrespect to the negative control) of the animals immunized with thevariants pMAE5Δ5-VEGF₁₂₁, pMAE5Δ5-VEGF₁₆₅, pMAE5Δ5-VEGF₁₈₉, and pMAE5Δ5VEGF_(KDR(−)), at doses of 50 and 100 μg per animal (Table 4).

TABLE 4 Tumor volume and survival in mice immunized with differentvariants of naked DNA containing the different isoforms of the VEGF geneand a mutated variant. [DNA Tumor Vol. (mm³) Survival Group μg] (Day 24)(Day 43) PAECΔ2-VEGF₁₂₁ 100 991.5 ± 354 (*) (ns) 50 1429.2 ± 396 (ns)(ns) 10 1506.6 ± 442 (ns) (ns) 1 1660.5 ± 456 (ns) (ns) PMAE5Δ5-VEGF₁₂₁100 645.0 ± 215 (***) (***) 50 850.1 ± 463 (***) (***) 10 992.1 ± 410(*) (ns) 1 1560.3 ± 598 (ns) (ns) PMAE5Δ5-VEGF₁₆₅ 100 799.2 ± 335 (***)(***) 50 916.6 ± 390 (**) (**) 10 1000.5 ± 662 (*) (ns) 1 1845.3 ± 450(ns) (ns) PMAE5Δ5-VEGF₁₈₉ 100 790.1 ± 235 (***) (***) 50 996.5 ± 255 (*)(**) 10 1050.2 ± 362 (*) (ns) 1 1670.2 ± 408 (ns) (ns) pMAE5Δ5VEGF_(KDR(−)) 100 550.1 ± 335 (***) (***) 50 894.7 ± 408 (**) (***) 10991.8 ± 362 (*) (ns) 1 1489.3 ± 510 (ns) (ns) PBS pH 7.2 0 1673.9 ± 712Note: Tumor volume is reported as mean ± standard deviation (SD) of themeasures performed on the animals of each group, statistical comparisonswere performed using one-way ANOVA and a Bonferroni post-test. In thecase of survival, the reported statistical significance was obtainedusing the log-rank test to compare each group with respect to thecontrol group, in the indicated day. Statistical signification isindicated as (ns) p ≦ 0.05 non-significant; (*) p ≦ 0.05; (**) p ≦ 0.01;and (***) p ≦ 0.001.

Example 5 In Vivo Protection Experiments Through Immunization withpMAE5Δ5-VEGF₁₂₁ and pMAE5Δ5-KDR 1-3, in a Model of Collagen-InducedArthritis

Groups of 20 mice were vaccinated or not with the following variants:

1. pMAE5Δ5-VEGF₁₂₁ (50 μg of DNA/mouse) in PBS pH 7.22. pMAE5Δ5-KDR 1-3 (50 μg of DNA/mouse) in PBS pH 7.23. PBS pH 7.2 (Negative control)

In all cases immunization (day 0) was by im. route in the rear left footwith a total volume of 50 μl. All the animals were re-immunized 15 dayslater using the original immunization regime.

On day 5 the induction of autoimmune arthritis began by immunizationwith chicken collagen type II (Sigma), a model previously described byCampbell et al. (Campbell I K et al Eur. J. Immunol. 30: 1568, 2000).This immunization was repeated on day 26. The four extremities of eachmouse were evaluated on a daily basis according to the arthritis indexthat establishes punctuation from 0 to 3 for each limb due to thepresence in the examination of signs of erythema (1), inflammation (2),or articular rigidity (3), with a maximal value of 12. Mice started toshow clinical symptoms of arthritis development 23 days after theinduction, with the higher incidences at 50 days. Table 5 shows theanalysis of arthritis incidence in the animals of the differentexperimental groups. In days 40 and 55 a significant reduction onarthritis incidence was observed in vaccinated groups (1 and 2) ascompared to control group.

TABLE 5 Incidence of arthritis on selected days (40 and 55). GroupIncidence day 40 Incidence day 55 1 20/8 (40%) 20/9 (45%) 2 20/6 (30%)20/12 (60%) 3 20/10 (50%) 20/14 (70%)

Example 6 In Vivo Antiangiogenic Effect of Vaccination

Groups of 15 mice were vaccinated or not with the following variants:

1. pMAE5Δ5-VEGF₁₂₁ (50 μg of DNA/mouse) in PBS pH 7.22. pMAE5Δ5-KDR 1-3 (50 μg of DNA/mouse) in PBS pH 7.23. pMAE5 KDR C (50 μg/mouse) in PBS pH 7.24. PBS pH 7.2 (Negative control)

In every case, C57BI/6 mice were immunized by im. injection in the rearleft foot with a total volume of 50 μl. All the animals werere-immunized 15 days later using the original immunization regime.Thirty days after the last immunization, the in vivo angiogenesis wasevaluated in the animals using matrigel as described by Coughlin M C etal. (Coughlin M C et al. J. Clin. Invest. 101:1441, 1998). The animalspreviously vaccinated were divided in groups of 5 and injectedsubcutaneously in the abdominal middle line with 500 μl of matrigel(Becton Dickinson and Co., Franklin Lakes, N.J., USA) containing:

1. VEGF 50 ng/mL, Heparin 50 U/mL2. 10⁵ cells of B16-F10 melanoma

3. PBS

Six days later the animals were sacrificed and the matrigel plug wasextracted. Hemoglobin contents in the plugs were analyzed according tothe manufacturer instructions (Drabkin's reagent kit; Sigma DiagnosticsCo., St. Louis, Mo., USA). Vaccination with the plasmids coding for VEGFor its receptor KDR inhibit significantly (p<0.001) the VEGF inducedvascularization, as well as that induced by systems that are morecomplex: tumor cells.

Example 7 Obtainment of an Immunogen Based in the Non-Covalent Bindingof pMAE5Δ5-VEGF₁₂₁ to Different Adjuvant Agents

Different immunostimulatory agents, previously reported, were used,mixed with the pMAE5Δ5-VEGF₁₂₁ construction following with themethodology described below. The Opc protein from the outer membrane ofNeisseria meningitidis was purified according to the report of Musacchioet al. (Musacchio A et al. Vaccine, 67:751, 1997). 50 μg/mL ofpMAE5Δ5-VEGF₁₂₁ were added to 10 μg/mL of Opc with gentle shaking atacid pH. The resulting complex was extensively dialyzed overnight inendo-free PBS pH 7.2 (Sigma). The level of Opc protein-plasmid DNAassociation (Opc-pMAE5Δ5-VEGF₁₂₁) was checked by DNA visualization using1% agarose gel. More than 50% of the plasmid DNA was associated to theOpc protein.

Very small particles (VSSP) coming from complex of outer membraneproteins (OMPC) of Neisseria meningitides, supplied by the Center ofMolecular Immunology (R. Perez et al. U.S. Pat. Nos. 5,788,985, and6,149,921), were used for combination with the plasmid DNA of interest.VSSP (1 mg) were incubated with 5 mg of pMAE5Δ5-VEGF₁₂₁ overnight withgentle agitation. The resulting material was extensively dialyzed inendo-free PBS pH 7.2 (Sigma). The level of VSSP-plasmid DNA association(VSSP-pMAE5Δ5-VEGF₁₂₁) was checked by DNA visualization using 1% agarosegel. More than 50% of the plasmid DNA was associated to the VSSPparticles.

The Hepatitis C and Hepatitis B core particulated antigens (HCcAg andHBcAg) were produced according to a previous report (Lorenzo L J et al.,Biochem Biophys Res Commun 281:962, 2001). One mg of the antigens weremixed with 5 mg of the plasmid in an overnight incubation. The levels ofHCcAg or HBcAg-plasmid DNA association (HCcAg-pMAE5Δ5-VEGF₁₂₁, andHBcAg-pMAE5Δ5-VEGF₁₂₁, respectively) were checked by DNA visualizationusing 1% agarose gel. More than 50% of the DNA was associated to theantigenic particle, in each case.

Example 8 Experiments of In Vivo Protection with the pMAE5Δ5-VEGF₁₂₁Construction and Immune Response Adjuvants

Groups of 10 C57BL6 mice were vaccinated or not with the followingvariants:

1. pMAE5Δ5-VEGF₁₂₁ (1, 10 and 50 μg of DNA/mouse) in PBS pH 7.2

2. Opc-pMAE5Δ5-VEGF₁₂₁ (1, 10 and 50 μg of DNA/mouse) 3.VSSP-pMAE5Δ5-VEGF₁₂₁ (1, 10 and 50 μg of DNA/mouse) 4.HBcAg-pMAE5Δ5-VEGF₁₂₁ (1, 10 and 50 μg of DNA/mouse) 5.HCcAg-pMAE5Δ5-VEGF₁₂₁ (1, 10 and 50 μg of DNA/mouse)

6. PBS pH 7.2 (Negative control for group 1)7. Opc (Negative control for group 2)8. VSSP (Negative control for group 3)9. HBcAg (Negative control for group 4)10. HCcAg (Negative control for group 5)

Immunization procedures, as well as tumor challenge and tumor volumemeasurements were similar to those described in the previous example.The vaccine variants with doses similar or higher to 10 μg of DNA/mousedecreased tumor growth in comparison to the respective negative controls(Table 6). A significant superior survival as compared to that of therespective control, was observed for the animals immunized with the VEGFgene, associated or not with Opc, VSSP, HCcAg and HBcAg, asimmunopotentiator vehicles. All the variants with vehicle showed asignificant superior survival versus the respective control, for dosesstarting with 10 μg/mouse, while the naked DNA variant with the vectorpMAE5Δ5-VEGF₁₂₁, resulted significantly different from the negativecontrol at the dose of 50 μg/mouse (Table 6).

TABLE 6 Tumor volume and survival of mice immunized using differentimmunostimulatory agents. [DNA Tumor Vol. (mm³). Survival Group μg] (Day24) (Day 43) pMAE5Δ5-VEGF 50 1050.9 ± 689 (**) (ns) 10 1229.0 ± 596 (*)(ns) 1 1895.3 ± 596 (ns) (ns) OpC-pMAE5Δ5-VEGF 50 960.6 ± 456 (**) (**)10 1100.5 ± 615 (**) (*) 1 1654.8 ± 663 (ns) (ns) VSSP-pMAE5Δ5-VEGF 50884.6 ± 410 (***) (**) 10 1002.3 ± 598 (**) (*) 1 1532.7 ± 745 (ns) (ns)HBcAg-pMAE5Δ5-VEGF 50 950.1 ± 570 (**) (**) 10 1230.5 ± 662 (*) (*) 11867.2 ± 652 (ns) (ns) HCcAg-pMAE5Δ5-VEGF 50 950.1 ± 570 (**) (**) 101230.5 ± 662 (*) (*) 1 1867.2 ± 652 (ns) (ns) OpC (5 μg/mouse/dose) 5 μg2059.0 ± 687 (ns) (ns) VSSP 2156.0 ± 759 (ns) (ns) HBcAg (5μg/mouse/dose) 1998.2 ± 798 (ns) (ns) HCcAg (5 μg/mouse/dose) 1897.0 ±812 (ns) (ns) PBS pH 7.2 2073.0 ± 816 (ns) (ns) Note: Tumor volume isreported as mean ± standard deviation (SD) of the measures performed onthe animals of each group, statistical comparisons were performed usingone-way ANOVA and a Bonferroni post-test. In the case of survival, thereported statistical significance was obtained using the log-rank testto compare each group with respect to the control group, in theindicated day. Statistical signification is indicated as (ns) p ≦ 0.05non-significant; (*) p ≦ 0.05; (**) p ≦ 0.01; and (***) p ≦ 0.001.

Example 9 In Vivo Protection Experiment Using VEGF in its Protein Form

Groups 10 C57BL6 mice were vaccinated or not with the followingvariants:

VEGF165 (20 μg/mouse) with Complete and Incomplete Freund adjuvantComplete and Incomplete Freund adjuvant (negative control)

VEGF₁₆₅ antigen was obtained from a commercial source (SIGMA) with morethan 97% purity. Mice were immunized by the intraperitoneal route usingComplete Freund's adjuvant (Sigma) with re-immunizations in days 15 and30 by the same route but using Incomplete Freund's adjuvant. Tumorchallenge, and measurements of tumor volume were similar to thosedescribed in the previous example.

A significant reduction in tumor volume and increase survival wereobserved in the VEGF immunized group as compared to the controlnon-immunized group. The effect was similar to those found in previousexperiments using VEGF DNA.

Example 10 In Vivo Experiments of Immune Protection Transfer in C57BL/6Mice with Severe Combined Immunodeficiency (SCID)

C57BL6 mice were immunized or not with doses of 50 μg of pMAE5Δ5-VEGF₁₂₁DNA/mouse using the procedures described in the example 5. Mice weresacrificed at 45 days after first immunization. CD8+, CD4+ andB-lymphocytes of these mice were separated using magnetic beads(Dynabeads, USA), according to the manufacturer instructions.

Groups of 10 six-week old C57BL6 SCID mice were reconstituted with thefollowing combinations of the previously extracted lymphocytes.

Group 1: CD8+ T-lymphocytes and CD4+ T-lymphocytes from mice immunizedwith pMAE5Δ5-VEGF₁₂₁ DNA. B-lymphocytes were not reconstituted.

Group 2: B-lymphocytes and CD4+ T-lymphocytes from immunized mice, andCD8+ T-lymphocytes from non-immunized mice.

Group 3: B-lymphocytes, CD8+ T-lymphocytes and CD4+ T-lymphocytes fromimmunized mice, as a positive control of the experiment.

Group 4: B-lymphocytes, CD8+ T-lymphocytes, and CD4+ T-lymphocytes fromnon-immunized mice, as a negative control of the experiment.

Reconstituted SCID mice were challenged sc. with 10⁴ B16-F10 melanomacells. Tumor growth was monitored by three weekly measurements untilmice start to die. Anti-VEGF antibody levels were analyzed through alaboratory ELISA. 96-well plates were incubated overnight with a 0.5μg/ml solution of VEGF165 (Sigma). The wells were blocked with PBS-BSA1% (BDH, UK) solution, and later incubated with serial dilutions of theanimal sera. After washing with PBS-Tween 0.05%, a commerciallyavailable polyclonal anti mouse IgG (Sigma, A0168) was added. The signalwas amplified in the presence of the commercial substrateortho-phenilene-diamine (OPD, Sigma).

Table 7 reflects the results of tumor volume (Day 24) and survival (Day40) of the groups of mice submitted to tumor challenge. Beginning on theday 15 after reconstitution, the animals of the groups 1 to 3experienced a reduction in tumor size as compared to group 4,reconstituted with lymphocytes from non-immunized mice. Thus, the effectthat provokes the immune system in the immunized mice, that allows thereduction in tumor size, is related to humoral and cellular responses,being the last one of the cytotoxic type (CTL), due to the absence ofanti-VEGF antibodies in group 1. Nevertheless, in the experimentalconditions used survival only increased in group 3 (B and T lymphocytesof immunized mice), as compared to the rest of the groups (Table 7). Inthe partially reconstituted animals where B or T of the CTL typeresponses were absent (groups 1 and 2, respectively) the survival wasnot different from the negative control. These results demonstrate thatthe combination of humoral and cellular responses (group 4), have asynergic effect that enables an effective response able to prolong thesurvival of mice submitted to the tumor challenge.

TABLE 7 Tumor volume and survival in SCID mice reconstituted withlymphocytes from pMAE5Δ5-VEGF₁₂₁ immunized mice. Mice donatinglymphocytes to the C57BL/6 SCID Tumor Vol. Survival Group B Lymph. CD4+Lymph. CD8+ Lymph. (Day 24) (Day 40) 1 — immunized immunized 1067.8 ±689 (ns) (ns) 2 immunized immunized non immunized 1129.0 ± 596 (ns) (ns)3 immunized immunized immunized  652.3 ± 396 (***) (***) 4 Non immunizedNon immunized Non immunized 1856.0 ± 756 — Note: Donor mice wereimmunized or not with doses of 50 μg of pMAE5Δ5-VEGF DNA per mouse.Tumor volume is reported as mean ± standard deviation (SD) of themeasures performed on the animals of each group, statistical comparisonswere performed using one-way ANOVA and a Bonferroni post-test. In thecase of survival, the reported statistical significance was obtainedusing the log-rank test to compare each group with respect to thecontrol group, in the indicated day. Statistical signification isindicated as (ns) p ≦ 0.05 non-significant; (*) p ≦ 0.05; (**) p ≦ 0.01;and (***) p ≦ 0.001.

Example 11 Demonstration of Immunological Restoration by Depletion ofCirculant VEGF Through Immune Response

Groups of 15 C57BL6 female mice were injected by im. route with thefollowing variants:

1. pMAE5Δ5-VEGF₁₂₁ (50 μg/mouse) in PBS pH 7.2

2. PBS pH 7.2

In every case, mice were immunized by im. injection in the rear leftfoot with a total volume of 50 μl. All the animals were re-immunized 15days later using the original immunization regime. Thirty days after thelast immunization 5 randomly selected animals from each group weresacrificed to analyze the immunological state of the immunized andcontrol animals, as well as the toxicity of vaccination on organs andtissues, through macroscopic and histological evaluations.

Remaining animals of each group received a sc injection of 10⁴ melanomaB16-F10 cells in the right ventral zone. At 15 and 30 days after tumorcells injection, 5 mice per group were sacrificed and evaluated aspreviously described.

Toxic events were not evidenced at macroscopic level in any of theevaluated animals, and histopathological analysis reveal no damage inany of the organs examined 30 days after the last immunization.Immunological evaluation consisted of: (1) evaluation of murine VEGFlevels in serum; (2) cellular content of T and B lymphocytes, as well asthe degree of maturity of dendritic cells in spleen, and in brachialaxillary and inguinal lymph nodes.

The analysis of the levels of murine VEGF (R&D kit for murine VEGF) inthe sera of un-treated animals showed that with the increase of time ofexposal to tumor, the VEGF levels increased in serum, in concordancewith the increase of tumor size with time. In the group immunizedagainst human VEGF a significant reduction (p<0.001 ANOVA, post-testBonferroni) of murine VEGF levels was detected, that lasted past 30 daysafter the tumor challenge.

The status of the immune system of the animals sacrificed on each momentwas analyzed through the study of the proportions of the cellularpopulations present on lymph nodes and spleen, according to the reportsof Gabrilovich et al. (Gabrilovich D et al. Blood 92:4150, 1998). Fortheses studies, commercial monoclonal antibodies that recognize CD3,CD19, CD11c and CD86 (B7-2) molecules (Pharmingen) labeled withfluorescein isothiocyanate (FITC) and phycoerythrine (PE), were used,that allowed the visualization of the cellular populations using a flowcytometer (FACS). Results obtained are shown in table 8.

TABLE 8 Summary of the results of FACS analysis of cell populationsaccording to surface markers. Fraction enriched with dendritic Total ofCells cells Lymph Nodes Spleen Lymph Nodes Spleen Group (day) CD-19 CD-3CD-19 CD-3 CD-11C/B7-2 CD-11C/B7-2 A. Non immunized Non immunized   8%  86% 38.1% 40.8%   60% 62.4% (30 Days) After tumor 20.1% 60.5% 3.811.4% 32.8% 10.2% challenge (60 Days) B. Immunized Immunized  7.2% 87.3%40%   39% 58.6% 60.3% (30 Days) After tumor 10.9% 80.1% 25.4   34% 53.5%52.9% challenge (60 Days) Note: In every case, values indicate thepercent of positive cells from the total of quantified cells.

The analyses of lymphoid cell populations and of the maturation ofdendritic cells in the animals, 30 days after the immunization, indicatethat the vaccination with the VEGF DNA does not induce any change in theimmune status of the animal. Nevertheless, 30 days after the tumorimplantation, the non-vaccinated animals show a decrease in theT-lymphocyte/B-lymphocyte ratio (CD3/CD19) both in lymph nodes and inspleen, with respect to the ratio before the tumor challenge.Furthermore, in particular in the spleen, there is a significantreduction in the number of lymphoid cells. A reduction in the number ofmature dendritc cells both in lymph nodes and in spleen was alsoobserved in these animals. In the group of mice vaccinated with the VEGFDNA a significant recovery in all parameters was evidenced, that couldbe correlated with the reduction of the VEGF levels in the sera observedin the animals of this group.

1.-33. (canceled)
 34. An immunogenic composition comprising a fusionprotein containing a VEGFR2 or fragments thereof and a mutant of VEGF,administered in the presence of or incorporated into Neisseriameningitidis outer membrane derived VSSP. 35.-79. (canceled)